Omega-3 fatty acid nutriceutical composition and optimization method

ABSTRACT

A novel omega-3 fatty acid/lipid based nutraceutical composition and a method of optimizing said omega-3 fatty acid/lipid based nutraceutical composition. The nutraceutical composition and method is based on the insight that different forms of high omega-3 fatty acid lipids (e.g. triglyceride form, ethyl ester form, free fatty acid form, phospholipid form) have different molecular modes and levels of action. Specifically the phospholipid form is likely more effective at promoting membrane fluidity and permeability, while the free fatty acid form is likely more effective at regulating cell receptors, such as the PPARα receptors, that are responsible for various metabolic effects including lipid metabolism. The desirability of producing omega-3 compositions that may act synergistically and thus more robustly to improve health and to some extent mimic markers of life extension such as shown by caloric restriction, along with specific optimization methods, markers, and compositions are taught.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of nutriceuticals and nutritionalsupplements, as well as methods of optimizing the composition ofnutriceuticals and nutritional supplements.

2. Description of the Related Art

Omega-3 Fatty Acids:

Omega-3 fatty acids, also called n-3 poly unsaturated fatty acids(PUFA), have long been suspected of having beneficial effects in humans,particularly with regards to reducing the risk of coronary heartdisease, reducing obesity, improving diabetic parameters including bloodglucose levels, and improving other parameters of the metabolicsyndrome. These fatty acids have a number of beneficial effects, amongwhich is lowering elevated blood triglyceride levels down to moreclinically acceptable values (Harris et. al., “Omega-3 fatty acids andcoronary heart disease risk: Clinical and mechanistic perspectives”Atherosclerosis. 2008 March; 197(1): 12-24). Omega-3 fatty acids canalso assist in weight/fat loss in overweight individuals.

Humans, and indeed essentially all animals, do not synthesize omega-3fatty acids directly. Instead these fatty acids must be obtained fromthe diet and therefore are essential dietary nutrients. These fattyacids present in relatively high levels in cold water fish, and othercold water marine animals such as Antarctic hill. The fish themselves donot synthesize the omega-3 fatty acids either, but rather acquire them,usually ultimately from phytoplankton, by virtue of the fish's positionon the marine food chain. The commercial omega-3 fatty acids arepurified from these marine sources, often by a molecular distillationprocess to remove unwanted impurities such as mercury, and are sold inboth over-the-counter and prescription forms as “fish oil”.

The structures of the various omega-3 fatty acids have been reviewed byRustan and Devon, “Fatty Acids: Structures and Properties” Encyclopediaof Life Sciences (2001). As they discuss, the most common animal fattyacids can be considered to be carbon chains (typically with a lengthbetween about 12 to 22 carbons, and often between 18 and 22 carbonslong) with one end terminating at a methyl group, and the other endterminating at a carboxyl group.

The saturated fatty acids are all composed of single carbon-carbonbonds, with the rest of the bonds being primarily occupied withhydrogen, while the unsaturated fatty acids have carbon-carbon doublebonds at various positions. The positions of the various carbon atoms inthe fatty acid chains are numbered with respect to the terminal methylgroup, and from a health standpoint, some of the more importantunsaturated fatty acids have double bonds starting between the carbon 3and carbon 4, and are these called n-3 or omega-3 fatty acids. Otherimportant fatty acids have double bonds starting between the carbon 6and carbon 7 on the chain, and these are called n-6 or omega-6 fattyacids.

Omega-3 fatty acids with a chain length of 20 atoms are calledeicosapentaenoic acid (EPA), while omega-3 fatty acids with a chainlength of 22 atoms are called docosahexaenoic acid (DHA). EPA is theprecursor for a number of different hormone-like molecules, such as theprostaglandins, and it also has an impact on platelet aggregation. EPAand DHA, when incorporated into cell membranes, are also known toincrease membrane fluidity, which may make it easier for blood tocirculate, and which may be at least partially responsible for theirpositive effect on cardiovascular health.

Because it is also cumbersome to repeatedly refer to the EPA and DHAform of the omega-3 fatty acids, in general when the term omega-3 fattyacids are used, the term should also be construed as covering at leastthe most common EPA and DHA forms of these fatty acids.

In addition to marine sources, certain land plants, such as flax (flaxseeds), walnuts, and the like also synthesize omega-3 fatty acids aswell, but they often synthesize these omega-3 fatty acids in the form ofshorter carbon chains (e.g. 18 carbons), such as alpha-linolenic acid(ALA). Although humans and animals can convert the ALA form to the moreuseful EPA and DHA form, the process is not particularly fast orefficient, and ALA is generally regarded as being a less favorableomega-3 fatty acid, while EPA and DHA, which do not require chain lengthconversion, are regarded as being more favorable.

The vast majority of the omega-3 fatty acids naturally occurring in fishoil are primarily present in a triglyceride form in which three fattyacid molecules, one or more of which can be an omega-3 fatty acid, andsome of which may be non-omega-3 fatty acids, are bound to a threecarbon glycerol backbone. This triglyceride form of the omega-3 fattyacids is the same form that is naturally used by the body to transportthe omega-3 fatty acids in the blood circulation.

Note that the word “triglycerides” is commonly used in the field to bothdescribe a particular form of omega-3 fatty acid composition, and alsoto describe blood fats in general, of which a positive effect of theomega-3 fatty acids is to lower the level of (non-omega-3) triglyceridesthat are circulating in the blood. To avoid confusion, “bloodtriglycerides” will be used to refer to the medical effect of theomega-3 fatty acids.

Although omega-3 fatty acids are predominantly found in unprocessed fishin a triglyceride form, natural, unprocessed fish oil is not generallyused directly as a nutritional supplement due to safety concernsregarding unwanted environmental contaminants such as mercury. To avoidthese unwanted contaminants, fish oil processors generally employmolecular distillation techniques to purify and/or concentrate the fishoil.

As a side effect of this molecular distillation process, most of thetriglyceride form of the omega-3 fatty acids are converted to asynthetic (not-found in nature) ethyl ester form for encapsulation orbottling. That is, in the purification and concentration process, thevarious omega-3 fatty acid residue(s) are severed from the glycerolbackbone of the triglyceride, and as a result of the process generallyused, an artificial ethyl ester form of the omega-3 fatty acid isgenerated.

In the ethyl ester form, the terminal —OH group of the omega-3 fattyacid head carboxyl group is replaced by an ethyl alcohol —OH—CH2-CH3group. This modification is considered harmless because once ingested;the user's liver can then, at least eventually, subsequently remove thesynthetic alcohol group from the omega-3 fatty acid ethyl ester. Howeverthis conversion is neither 100% efficient nor instantaneous.

Alternatively, the manufacturer can, at a higher expense, take theethyl-ester form of the omega-3 fatty acids, as well as glycerol andother fatty acids, and recreate an artificial omega-3 fatty acid in atriglyceride form that, to all intents and purposes is equivalent to theoriginal natural omega-3 fatty acid in triglyceride form. NordicNaturals, of Watson Calif., for example, produces their “Ultimate Omega”line of fish oil products using this approach. Due to the higher expenseof this extra process, however, this approach is less common.

Because the ethyl ester form of the omega-3 fatty acids is cheaper toproduce, and because manufacturers are understandably reluctant topublicize that they are providing an unnatural form of omega-3 in theirnutritional supplements, unless fish oil label clearly says otherwise,it should be assumed that the fish oil contains a high proportion of theomega-3 fatty acids in the ethyl ester form.

Natural fish oil supplements thus generally consist of either thetriglyceride form of EPA and DHA form of omega-3 fatty acids, or thesynthetic ethyl ester form of EPA and DHA omega-3 fatty acids.

In addition to the natural triglyceride form of omega-3 fatty acids, thenatural free fatty acid form of omega-3 fatty acids, and the artificialethyl ester form of omega-3 fatty acids, another form of omega-3 fattyacids also exists, the phospholipid form.

The phospholipid form of omega-3 fatty acids is found in highconcentrations in certain marine animal species adapted for life inextremely cold water, such as Antarctic hill (e.g. Euphausia superba).In this form, at least one omega-3 fatty acid residue, often inconjunction with a non-omega-3 fatty acid residue, is again attached toa glycerol backbone, but the third position on the glycerol backbone isoccupied by a phosphate group, which in turn is usually conjugated witha choline, serine, or ethanolamine group. Such phospholipid forms of theomega-3 fatty acids likely help the krill continue to function atextremely low temperatures because they help keep the hill cellmembranes fluid. In this respect, the phospholipid forms of the omega-3fatty acids can somewhat considered to be acting like a natural versionof a cell membrane antifreeze.

Thus in contrast to standard fish oil, the omega-3 fatty acids inpurified Antarctic hill oil supplements generally consists of a mix ofaround 40%-50% phospholipids (which has omega-3 EPA and DHA carbonchains), and the rest of the omega-3 fatty acid groups are generallyfound either in the triglyceride form (natural form before moleculardistillation) or in the ethyl ester form (after molecular distillation).

Although fish oil has long been a favorite of the nutritional supplementindustry in various over-the-counter (non-prescription) forms, recently,the therapeutic utility of the omega-3 fatty acids has also attractedthe attention of various pharmaceutical companies. For example, Lovaza,produced by GalaxoSmithKline, is a purified ethyl ester form of theomega-3 fatty acids EPA and DHA. Similarly Epanova, produced by OmtheraPharmaceuticals, is a purified free fatty acid form of the omega-3 fattyacids.

PPAR Receptors:

The omega-3 fatty acids are believed to mediate some of their actions,at least in part, by way of the peroxisome proliferator-activatedreceptors (PPARs). PPARs are nuclear receptor proteins that bind toretinoid hormones (e.g. hormones built around a carbon chain backbonethat is somewhat similar to the carbon chain backbone of omega-3 fattyacids, such as prostaglandins, vitamin D, and relevant to thisdiscussion, also omega-3 fatty acids in the non-esterified, free fattyacid form).

The PPAR receptor proteins are a family of proteins that exist invarious forms, called the α, β, δ, γ1, γ2, γ3 form. After a PPARreceptor binds its particular ligand, it then forms a dimer with aretinoid Z receptor, and this complex in turn binds to the DNA ofvarious genes, thereby regulating the transcription of these genes.

The PPARα (alpha) receptors, for example, are primarily expressed in theliver and fat cells (adipose tissues), and play a critical role in bothfat metabolism and diabetes. The PPARγ (gamma) receptors are alsoexpressed in adipose tissues as well. Various drugs involved inregulating both triglyceride production and diabetes target variousmembers of the PPAR family. For example, fibrate blood triglyceridelowering drugs target PPARα receptors. By contrast, variousanti-diabetic drugs, such as the various thiazolidinediones (exemplifiedby drugs such as Avandia and Actos) target the PPARγ receptors.

Mutations in PPAR receptors have been linked to lipid disorders, insulinresistance, and obesity.

As previously discussed, prior studies have suggested that the omega-3or n-3 fatty acids may themselves interact with some PPAR receptors. Forexample, Jump, in “Dietary polyunsaturated fatty acids and regulation ofgene transcription”, Curr. Opin. Lipidol. 2002 April; 13(2):155-64,reviewed various studies showing that non-esterfied (e.g. free, unboundto glycerol) fatty acids or fatty acid metabolites, in particular 18 and20 carbon long fatty acids which are n-3 polyunsaturated fatty acids,may activate PPAR receptors, in particular PPARα

PPARα (PPAR-alpha) is a transcription factor and a major regulator oflipid metabolism in the liver and other organs. Activation of PPARαpromotes uptake, utilization, and catabolism of fatty acids byup-regulation and expression of genes involved in fatty acid transportand peroxisomal and mitochondrial fatty acid β-oxidation. PPARα isprimarily activated through ligand binding such as by the free fattyacid form of omega-3 fatty acids.

It should be noted that when PPARα is stimulated, it changes the cell'sgenetic expression and metabolism to an alternate state which betterenables fats to be burned as fuel. On the other hand, PPARγ (PPAR-gamma)stimulation, as by current diabetic pharmaceuticals, results in theinhibition of fat burning, increased fat storage, and the manufacture ofnew fat cells. This effect of PPARγ is obviously less than desirable forthe majority of diabetics and the many overweight people throughout theworld. Effective stimulation of PPARα for instance could, on the otherhand, present powerful and beneficial benefits.

Caloric Restriction:

Another technique of regulating many metabolic activities in a favorabledirection is caloric restriction. Caloric restriction has widely beenrecognized for over 70 years as being an effective means of prolongingmaximal lifespan in many species, including mammals such as rodents, andeven primates. As a result, some human enthusiasts have embraced this asa form of life extension protocol. However this life extension protocol,although possibly effective, is very hard to follow.

Caloric restriction diets, which are not generally consideredappropriate for individuals under the age of 21, generally require humanpractitioners to eat between 10-25% fewer calories than average. Thesediets have been shown to produce impressive health results thus far inhumans, including a reduction in cardiovascular disease markers asindicated by LDL particle number and size, coronary artery imagingtechniques, carotid artery elasticity. Such diets also increase HDL,lower blood pressure, lower triglyceride levels, and are also associatedwith improved memory, and reduced inflammation. Because such diets arehard to follow on a long-term basis, however, there is great interest infinding methods to biochemically reproduce the desirable effects ofcaloric restriction without the hardships of caloric restriction.

Interestingly, recent work suggests that there may be a relationshipbetween caloric restriction and the PPAR receptors. For example, Corton,et. al., “Mimetics of Caloric Restriction Include Agonists ofLipid-activated Nuclear Receptors”, J. Biol. Chem. 279 (44), 46204-46212(2004) studied the impact of caloric restriction on normal and mutantPPARα-null mice. They found that the beneficial effects of caloricrestriction were lacking in the PPARα-null mice, suggesting that thePPARα receptors may play a role in mediating the beneficial effects ofcaloric restriction.

Membrane Fluidity

On a side note, note, but relevant to this invention, a brief review ofbiological membranes is also in order.

Biological membranes are composed of a bilayer of membrane lipids,primarily cholesterol, glycolipids, and phospholipids. The lipid bilayerstructure is thermodynamically favored because hydrophobic effects causethe lipid hydrocarbon chains to coalesce together to form an internalhydrophobic environment inside the membrane, while at the same time, thehydrophilic phosphate polar head groups of phospholipids face theexterior aqueous environment, thus creating a two dimensional lipidbilayer membrane structure.

Because the various lipid molecules are only weakly held into positionin the membrane by hydrogen bonds, they are to some extent, free to movearound within the two dimensional plane of the membrane. Thus biologicalmembranes act in some respect like a two dimensional fluid.

This fluidity is in fact an essential part of the proper biologicaloperation of cells and cell membranes, because it allows embedded cellmembrane proteins to move about in a two dimensional space and performvarious functions that would otherwise not be possible if they wereforced to be stationary.

The fluidity of a biological membrane varies, to some extent, dependingon both ambient temperature and lipid composition. At coolertemperatures, such as those experienced by cold blooded marine animals,phytoplankton, and also plants, membrane fluidity is generally much lessand more difficult to maintain than it would be in a warm blooded mammalenvironment.

Thermodynamically, what happens is that at lower temperatures, thehydrogen bonding between the different lipid molecules starts todominate over the thermodynamic fluctuations that would otherwise causethese bonds to break. To cope with this problem, cold environment plantsand marine animals incorporate a larger number of unsaturated omega-3fatty acid residues in their membrane lipids. The double bonds in theomega-3 fatty acids tend to disrupt or not participate in the hydrogenbonding between different lipids, and thus promote membrane fluidity andagain somewhat act like “anti-freeze” in this regard.

Absent specific transport mechanisms, such as transport proteins, pores,and the like, the cell membrane is generally fairly impermeable to mostmolecules, including free fatty acids, hormones, and the like. Howeverworkers, such as Lande et. al. Journal of General Physiology 106, 67-84(1995) al., have noted that at least for some classes of molecules,higher membrane fluidity is positively correlated with increasedpermeability. Generally increased fluidity is also likely to helpspecific transport proteins and membrane receptors function with higherefficiency such as the glucose transporter complex GLUT4 and the insulinreceptor, improving control of blood glucose and diabetes.

BRIEF SUMMARY OF THE INVENTION

The invention is based, in part, on the insight that information fromthe above teaching can be combined to create a model of omega-3 functionthat predicts that under proper conditions; the various forms (e.g. freefatty acid forms, phospholipid forms) of the omega-3 fatty acids can becombined in a way that produces synergistically favorable healtheffects. This new omega-3 function model is based on the premise thatcertain forms (e.g. phospholipid forms) of omega-3 fatty acids arebetter at enhancing membrane fluidity and permeability, while otherforms of omega-3 fatty acids (e.g. free fatty acid forms) are better atstimulating biological receptors of interest, such as the PPARαreceptors.

This new omega-3 function model in turn suggests both a new experimentalapproach for how to better optimize omega-3 fatty acid nutraceuticals,and also suggests new types of omega-3 nutraceutical compositions. Theinvention is further based on the insight that regardless of whether ornot the new omega-3 function model is correct all details, the noveloptimization methods and the novel omega-3 nutraceutical compositionshave independent value and validity.

The invention is further based, in part, on the insight that it would bedesirable to optimize the formulation of omega-3 oils, such as fish oil,to attempt, to the greatest extent possible, to duplicate at least someof the beneficial effects of caloric restriction as a model for improvedhealth.

The invention is also based, in part, on the insight that the newomega-3 function model predicts that prior art omega-3 fatty acidformulations, such as prior art fish oil and/or hill oil formulationsand the various pharmaceutical formulations, are not fully optimized.That is, none of the prior art formulations have been optimized for thepurpose of delivering the highest effective concentration of omega-3agonists to suitable health promoting and/or caloric restrictiontargets, such as the PPARα receptors.

The invention is also based, in part, on the insight that at least somehealth promoting/caloric restriction receptors, such as the PPARαreceptors, are nuclear receptors. Thus to deliver a proper agonistsignal to these nuclear receptors not only must the omega-3 be in a formthat the receptor recognizes, i.e. the free fatty acid form, but theomega-3 must additionally be in a form that improves membrane fluidityso as to help allow the free fatty acid form of omega-3 to penetrate atleast past the cell membrane and often the nuclear membrane as well, andthat form of omega-3 would best be as a phospholipid.

The invention is also based, in part, on the insight that an omega-3formulation optimized, on the one hand, to promote membrane fluidity bydelivering an effective amount of omega-3 phospholipid fatty acids, andoptimized on the other hand for high levels of PPARα (and otherreceptor) agonist activity by also delivering an effective amount of thefree fatty acid form of omega-3, would have a higher probability tomanifest beneficial health effects, such as the health effectsassociated with caloric restriction.

The invention is also based, in part, on the insight that for maximaleffectiveness, the forms of the omega-3 fatty acids that are providedshould be directly usable by the body in a manner that requires from thebody the fewest number of intermediate enzymatic processing steps. Thiswould be particularly useful because in many chronic disease states,such as obesity, diabetes and cardiovascular diseases, some of theseintermediate enzymatic processing steps may not operate with normalefficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the difference between the free fatty acid forms, ethylester forms, triglyceride forms, and phospholipid forms of an omega-3fatty acid such as EPA.

FIG. 2 shows a simplified diagram of the model of omega-3 function whichinspired both the invention's nutraceutical compositions andoptimization methods. Here the phospholipid forms of omega-3 fatty acidspromote membrane fluidity and omega-3 free fatty acid uptake, while thefree fatty acid form of omega-3 then serves (for example), as a PPARαreceptor agonist. In this model, the phospholipid form of omega-3 fattyacids and the free fatty acid form of omega-3 fatty acids thus actsynergistically, as the phospholipid form of omega-3 fatty acids makesit easier for the free fatty acid form of omega-3 fatty acids to reachthe membrane-protected PPARα receptors. Here Omega-3 is abbreviated asΩ3.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows the difference between the free fatty acid forms, ethylester forms, triglyceride forms, and phospholipid forms of an omega-3fatty acid such as EPA.

Here, the chemical structure of EPA (100) in the free fatty acid form,which is CH3CH2CH═CHCH2CH═CHCH2CH═CHCH2CH═CHCH2CH═CH(CH2)3COOH can beabbreviated as R1-COOH (102). Similarly the chemical structure of EPA inthe ethyl ester form can be abbreviated as R1-CO—OCH2CH3 (104).

EPA in the 3-chain glycerol form (106), assuming that only one of thechains R1 is EPA, and the other two chains may be different fatty acids,often common C16 and C18 fatty acids (R2 and R3) is:

By contrast, EPA in the phospholipid form (108) has the chemicalstructure:

where X may be a side group such as choline, serine, or ethanolamine

As previously discussed, manufacturers are usually reluctant to disclosethe fact that they have chosen to keep the processed omega-3 fatty acidsin their unnatural ethyl ester form. Since there appear to be nogovernment regulations requiring this level of detail to be disclosed,manufacturer labels are often very cryptic in this regard. Althoughcomparative levels of EPA and DHA are reported, the labels otherwiseusually fail to specify what form (triglyceride, free fatty acid, orethyl ester) the omega-3 fatty acids are in.

As a result, the terminology and literature in this field can be highlyconfusing, and citations must be read carefully. Usually the simple term“fatty acids” generally refers to the basic carbon chain backbone of themolecule(s), rather than the specific form of this basic carbon chainbackbone. To reiterate, when the omega-3 fatty acids are in the form ofthe carboxylic acid and also not attached or “esterified” to a glycerolbackbone as part of a triglyceride, they are termed “free fatty acids”.When the omega-3 fatty acids have an ethyl ester attached, they aretermed omega-3 acid ethyl ester”. Similarly the omega-3 fatty acidmolecule can also be attached to a 3 carbon glycerol backbone with twoother fatty acids (omega-3 or not) and form a three carbon chaintriglyceride, or the omega-3 fatty acid molecule can be attached to aglycerol backbone that has another fatty acid attached, along with aphosphate residue on the 3^(rd) carbon, and form a phospholipid.

The one partial exception to the ambiguity in disclosure is hill oil,where phospholipid forms of the omega-3 fatty acids are particularlyvalued. Here manufacturers usually at least report on the percentage ofthe oil that is in the phospholipid form. Here again, however,manufacturers will still typically fail to disclose the exact form ofthe remainder of the composition, or even specify how much of thephospholipid has EPA or DHA residues. Thus, although percentages ofphospholipids and percentage of EPA and DHA may be reported, thespecific forms of the EPA and DHA (percentage in triglyceride, ethylester, etc.) will again usually not be reported, or likely even known.Here again, the presumption should be that unless otherwise reported, ahigh proportion of the ethyl ester form should be assumed.

Indeed the few manufacturers, such as Nordic Naturals, who use naturaland more expensive triglyceride forms of EPA and DHA, usually announcethese alternative forms by very prominent labeling in order to drawattention to this fact.

FIG. 2 shows the proposed molecular mechanism that inspired theinvention's novel omega-3 fatty acid composition and optimizationmethod. In this model, the phospholipid forms of omega-3 fatty acidspromote membrane fluidity and omega-3 free fatty acid uptake. Bycontrast, the free fatty acid form of omega-3 then can serve, forexample, as agonists (stimulators) for PPARα receptors and/or othernuclear, cytoplasmic, or cell surface membrane receptors.

In this model, consider a cell, such as a liver cell (200). The cell hasa cell membrane (202) and a nuclear membrane (203). Here thephospholipid form of the omega 3 fatty acids (108) can be incorporated“as is” (i.e. with little or no enzymatic alteration by the body) intoboth the cell membrane (204) and the nuclear membrane (208), increasingboth membrane fluidity and membrane permeability (206), (210). As aresult, aided or synergized by this increased membrane fluidity andpermeability, the free fatty acid form of the omega-3 fatty acids (102)can now more effectively permeate or be transported across the cellmembranes (202) and (203) “as is”. Once inside the cytoplasm or nucleus,the free fatty acid form of the omega-3 fatty acids is now free to actas an agonist or stimulator of receptors, such as PPARα receptors (212).Once stimulated, the PPARα receptors (214) or other receptors can inturn mediate other cellular pathways. In this particular example, theactivated PPARα receptor (214) binds to one or more regions of thecellular DNA (216), and stimulates transcription of one or more genes(218).

This model suggests both that the various forms of the omega-3 fattyacids do not act by the same mechanism, and further that some forms,such as the triglyceride form of omega-3 fatty acids and the ethyl esterform of omega-3 fatty acids are possibly less effective at some of thesefunctions. In this model, for example, neither the triglyceride form northe ethyl ester form, “as is”, is envisioned as being as effective ateither stimulating membrane fluidity or stimulating receptors such asPPARα receptors. This model thus makes a prediction that thetriglyceride form of the omega-3 fatty acids and the ethyl ester form ofthe omega-3 fatty acids may be less favored for this type ofapplication. Furthermore, though the phospholipid form of omega-3 mightbe best at enhancing membrane fluidity and permeability, is not likelyto be best at stimulating receptors. Likewise, though the free fattyacid form of omega-3 fatty acids are likely the best form, and possiblythe only form, that can stimulate receptors such as PPARα, it is notlikely to be the form best at enhancing membrane fluidity, as this wouldrequire in vivo modification to the phospholipid form.

The limitations of the model should be appreciated, however. The modelhas value in that it predicts both a new optimization method and a newomega-3 nutraceutical combination. However since both the optimizationmethod and the new omega-3 nutraceutical combinations have valueindependent of the model itself, other omega-3 forms may also be studiedby the proposed optimization method and also explored as variousalternative omega-3 nutraceutical combinations. Thus, use of both thetriglyceride form and the ethyl ester form of omega-3 fatty acids is notdisclaimed, and indeed in some alternative formulations, positivesynergistic effects may potentially be seen with either the triglycerideform or the ethyl ester form as well.

Thus the model constitutes a valid scientific hypothesis which in turnsuggests both novel optimization methods and new compositions. Thus thespecific PPAR receptor teaching and membrane fluidity teaching discussedhere should be considered to be only one example of an aspect orembodiment of the invention, and is not intended to be limiting.

A table showing how the invention's omega-3 fatty acid nutraceuticalformulation contrasts with prior art prescription and over-the-counteromega-3 fatty acid nutraceutical formulations is shown in Table 1:

TABLE 1 Various forms of omega-3 fatty acids in prior- art omega-3 fattyacid formulations, versus the invention's omega-3 fatty acidformulation. omega-3 fatty acid forms: omega-3 omega-3 omega-3 omega-3free fatty ethyl triglyc- phospho- acids ester erides lipids Fish oil —— Nearly — (natural) 100% Fish oil — Nearly — — (molecular 100%distillation) Krill oil — — Roughly Roughly (natural)  50% 50% Krill oil— Roughly — Roughly (molecular  50% 50% distillation) Epanova Nearly — —— 100% Lovaza — Nearly — — (pharma- 100% ceutical grade moleculardistillation) Nordic Naturals — — Nearly (reconstituted 100%triglycerides) Invention 20-70% — — 20-70%

In one embodiment, the invention may be a nutraceutical dietarysupplement or food comprising the phospholipid form of omega-3 fattyacids (including EPA and DHA) and the free fatty acid form of omega-3fatty acids. In general, the phospholipid form of omega-3 fatty acidswill comprise between about 20 and 70 percent of the omega-3 fatty acidsin the nutraceutical supplement, the free fatty acid form of omega-3fatty acids will comprise between 20 and 70 percent of the omega-3 fattyacids in the nutraceutical supplement, the triglyceride form of omega-3fatty acids will comprise between 0 and 5% of the omega-3 fatty acids inthe nutraceutical supplement, and the ethyl ester form of the omega-3fatty acids will comprise between 0 and 5% of the omega-3 fatty acids inthe nutraceutical supplement.

In this supplement, the sum of all omega-3 fatty acids forms may also beequal to between 10 to 100% of the entire composition, and the remainderof the dietary supplement will be comprised of other materials such asother triglycerides or fatty acids such as medium chain triglycerides ormonounsaturated fats, antioxidants, emulsifiers (e.g. lecithin),carriers (e.g. gelatin, water), and the like.

Here, the specific amounts of the phospholipid form of the omega-3 fattyacids and the specific amounts of the free fatty acid forms of theomega-3 fatty acids may be adjusted so that the enhanced membranefluidity and permeability afforded by the omega 3 phospholipid form ofthe fatty acids results in greater proportions of the free fatty acidform of omega-3 fatty acids penetrating cellular membranes. Afterpenetration, the free fatty acid form of the omega-3 fatty acids maythen bind to the nuclear PPARα receptors for example, or other receptorsor molecular effectors such as genetic transcription factors, thuscreating an agonistic effect on these receptors and/or effectors. Inother words, the membrane fluidity and permeability enhancementproperties of the phospholipid form of the omega-3 fatty acids willsynergize with the PPARα or other receptor or other biochemical effectsof the free fatty acid form of the omega-3 fatty acids, thus creatingincreased PPARα (ανδ/or other) receptor activation (or other biochemicaleffects) than would be possible if either the phospholipid form or thefree fatty acid form of the omega-3 fatty acids were used separately.

Unsaturated fatty acids, such as the omega-3 fatty acids, oxidizerapidly in air, and often the nutraceutical supplement may also containvarious materials intended to retard oxidation, such as vitamin Etocopherols, tocotrienols, lipoic acid, astaxanthin, and other fatsoluble antioxidants. Additionally, the other materials may alsocomprise materials such as medium and short chain fatty acids, omega-6fatty acids, omega-9 fatty acids, choline, cholesterol, gelatin, andwater.

In general, the nutraceutical supplement may be formulated so that thepercentage of the phospholipid form of omega-3 fatty acids and thepercentage of the free fatty acid form of the omega-3 fatty acids arechosen based on their positive synergistic effect when taken by an adulthuman at a level of between about 1 gm and 30 gm (or two tablespoons) ofthe supplement on a daily basis.

The optimal levels of omega-3 phospholipid forms of the fatty acids andomega-3 free fatty acid forms may be determined by various means,including animal studies. Here, for example, the methods of Corton et.al. (Journal of Biological Chemistry 279 (44), 46204-46212 (2004) may beused. Test animals such as mice, or even human subjects, may be fed acontrolled diet containing various formulations of the nutritionalsupplement where the omega-3 fatty acids are set at various phospholipidto free fatty acid concentrations. The levels of gene expression(transcription) by the various lipid activated nuclear receptors, suchas the PPARα receptors, may then be monitored using standard methodssuch as reverse transcriptase-PCR methods as detailed by Corton. Thesegene transcription levels, which may be considered to be one type ofsurrogate endpoint associated with life extension, can then be analyzedversus the omega-3 phospholipid to omega-3 free fatty acid compositionof various nutritional supplement candidates, and the formulationassociated with the highest level of gene expression, such as thehighest level of PPARα activation, may be chosen.

In addition to looking directly at the transcription levels of certaingenes associated with life extension, other markers of life extensionmay also be monitored, and the levels of omega-3 phospholipid to omega-3free fatty acid associated with the desired effect (often the greatesteffect at which unwanted side effects that do not also occur) may bechosen. These can be surrogate endpoints associated with life extensionprotocols such as caloric restriction, and can include endpoints ormarkers associated with reduced free T3 levels, reduced fasting seruminsulin levels, reduced fasting serum leptin levels, reduced basal bodytemperature, reduced serum triglycerides, and enhanced beta fatty acidoxidation as indicated via a reduced respiratory quotient.

Although in principle, any positive synergistic effect produced by acombination of the phospholipid form and the free fatty acid form of theomega-3 fatty acids would be detected by the above methods, and would bealso quite acceptable in terms of the actual formulation. In particular,one synergistic effect that is expected according to the invention iswhere the synergistic effect is due to the free fatty acid form ofomega-3 fatty acids penetrating the cellular membranes at a higher ratedue to increased cell membrane permeability and/or fluidity induced bythe combined phospholipid form of the omega-3 fatty acids.

Also in principle, any positive synergistic effect on any agonisticeffect induced on any biochemical receptor (and/or other moleculareffector such as an enzyme or genetic transcription factor directly)would also be quite acceptable in terms of the actual formulation.However as previously discussed, in particular one synergistic effectmay be due to the phospholipid form of omega-3 lipid's promotion of theability of the free fatty acid form of omega-3 fatty acids to functionas a biochemical agonist for receptors and/or genetic transcriptionfactors, in particular as an agonist to PPAR nuclear receptors such asthe PPARα nuclear receptors.

Alternatively, the invention may be viewed as a method for optimizingthe composition of the omega-3 free fatty acids of a nutraceutical.

Although typically, the nutraceutical supplement will be delivered in apill form, often in the form of one or more pills that may deliverbetween 300 mg and 1000 mg of total oil per pill, of which often between25% to 100% of this oil may be the various forms of omega-3 fatty acidsdiscussed previously. However other forms of nutritional supplement mayalso be used. It may also be delivered as a bottled oil food orsupplement to be taken by spoonful. In alternative formulations, thenutritional supplement may be blended into other food products (e.g.peanut butter, margarine, salad oil, various drinks, and the like). Inother formulations, the nutritional supplement may be incorporated intovarious solid foods, or even delivered in a formulation suitable forenteric tube feeding or intravenous administration.

1. A nutraceutical comprising the phospholipid form of omega-3 fattyacids (including EPA and DHA) and the free fatty acid form of omega-3fatty acids; wherein the phospholipid form of the omega-3 fatty acidscomprise between 20 and 70 percent of the total omega-3 fatty acids, thefree fatty acid form of omega-3 fatty acids comprise between 20 and 70percent of the total omega-3 fatty acids, the triglyceride form ofomega-3 fatty acids comprise between 0 and 5% of the total omega-3 fattyacids, the ethyl ester form of omega-3 fatty acids comprise between 0and 5% of the total omega-3 fatty acids, and wherein the sum of allomega-3 fatty acids is equal to 100%, and the remainder of the dietarysupplement is comprised of other materials.
 2. The nutraceutical ofclaim 1, wherein said other materials comprise one or more materialsselected from the group consisting of vitamin E tocopherols,tocotrienols, alpha and/or other lipoic acids, astaxanthin, other fatsoluble antioxidants, CoQ10, 1-carnitine, acetyl 1-carnitine, medium andshort chain triglycerides, omega-9 fatty acids, lecithin, phosphatidylcholine, phosphatidyl serine, phosphatidyl ethanolamine, choline,cholesterol, gelatin, and water.
 3. The nutraceutical of claim 1,wherein said nutraceutical is provided in a pill form along withinstructions for use instructing that the nutraceutical should be takenby an adult human at a level of between 0.5 gm and 30 gm of saidnutraceutical on a daily basis.
 4. The nutraceutical of claim 1, whereinat least some of said phospholipid form of omega-3 fatty acids arederived from krill oil.
 5. The nutraceutical of claim 1, wherein atleast some of said phospholipid form of omega-3 fatty acids are derivedfrom algae, phytoplankton, or other vegetarian source.
 6. Thenutraceutical of claim 1, wherein at least some of said free fatty acidform of omega-3 fatty acids are derived from fish oil.
 7. Thenutraceutical of claim 1, wherein at least some of said free fatty acidform of omega-3 fatty acids are derived from algae, phytoplankton, orother vegetarian source.
 8. A nutraceutical comprising the phospholipidform of omega-3 fatty acids (including EPA and DHA) and the triglycerideform of omega-3 fatty acids; wherein the phospholipid form of omega-3fatty acids comprise between 20 and 70 percent of the total omega-3fatty acids, the triglyceride form of omega-3 fatty acids comprisebetween 20 and 70 percent of the total omega-3 fatty acids, the freefatty acid form of omega-3 fatty acids comprise between 0 and 5% of thetotal omega-3 fatty acids, the ethyl ester form of omega-3 fatty acidscomprise between 0 and 5% of the total omega-3 fatty acids; and whereinthe sum of all omega-3 fatty acids is equal to 100%, and the remainderof the dietary supplement is comprised of other materials.
 9. Thenutraceutical of claim 8, wherein said other materials comprise one ormore materials selected from the group consisting of vitamin E,tocopherols, tocotrienols lipoid acid, astaxanthin, other fat solubleantioxidants, CoQ10, 1-carnitine, acetyl 1-carnitine, medium and shortchain triglycerides, omega-9 fatty acids, choline, cholesterol, gelatin,and water.
 10. The nutraceutical of claim 8, wherein said nutraceuticalis provided in a pill form along with instructions for use instructingthat the nutraceutical should be taken by an adult human at a level ofbetween 0.5 gm and 30 gm of said nutraceutical on a daily basis.
 11. Thenutraceutical of claim 8, wherein at least some of said phospholipidform of omega-3 fatty acids are derived from Antarctic Krill oil. 12.The nutraceutical of claim 8, wherein at least some of said phospholipidform of omega-3 fatty acids are derived from algae, phytoplankton, orother vegetarian source.
 13. The nutraceutical of claim 8, wherein atleast some of said triglyceride form of omega-3 fatty acids are derivedfrom fish oil.
 14. The nutraceutical of claim 8, wherein at least someof said triglyceride form of omega-3 fatty acids are derived from algae,phytoplankton, or other vegetarian source.
 15. A method of optimizingthe relative composition and amounts of the various omega-3 fatty acidsof a nutraceutical composition, said method comprising; conducting aseries of controlled experiments with a plurality of experimentalgroups, each group composed of a plurality of experimental animals orhuman subjects; feeding a plurality of different nutraceuticalformulations composed of different omega-3 fatty acid forms to saidplurality of experimental groups; wherein each of said differentnutraceutical formulations has the same total amount of omega-3 fattyacids; monitoring said experimental groups for least one markerassociated with laboratory animal life extension; and determining whichof said plurality of different nutraceutical formulations optimizes saidat least one marker.
 16. The method of claim 15, wherein said differentomega-3 fatty acid forms comprise the triglyceride form of omega-3 fattyacids, the ethyl ester form of omega-3 fatty acids, the free fatty acidform of omega-3 fatty acids, and the phospholipid form of omega-3 fattyacids.
 17. The method of claim 15, wherein said different omega-3 fattyacid forms comprise the free fatty acid form of omega-3 fatty acids andthe phospholipid form of omega-3 fatty acids.
 18. The method of claim15, wherein the phospholipid form of the omega-3 fatty acids are chosento be at a level that enhances membrane fluidity and permeability;wherein the free fatty acid form of the omega-3 fatty acids are chosento be at a level capable of permeating said fluidity and permeabilityenhanced membranes and subsequently activating the PPARα nuclearreceptors and/or other nuclear receptors and/or molecular effectors suchas genetic transcription factors of their markers; and wherein therelative amounts of said phospholipid form of the omega-3 fatty acidsand free fatty acid form of the omega-3 fatty acids are chosen so as toprovide a synergistic level of nuclear receptor and/or moleculareffector activation that is greater than the level that would beobtained if the nutraceutical was either 100% phospholipid form ofomega-3 fatty acids or 100% free fatty acid form of omega-3 fatty acids.19. The method of claim 18, wherein the activation of the PPARαreceptors and/or other receptors and/or molecular effectors isdetermined by analyzing the level of gene transcription (via forinstance messenger RNA or their protein products) of the genes that areactivated or repressed by said activated PPARα receptors and/or otherreceptors and/or molecular effectors.
 20. The method of claim 15,wherein at least the percentage of the phospholipid form of omega-3fatty acids and the at least the percentage of the free fatty acid formof omega-3 fatty acids are chosen based on their positive synergisticeffect when taken by an adult human at a level of between 0.5 gm and 30gm (2 tablespoons) of said supplement on a daily basis or by anequivalent relative amount if laboratory animals are used in theexperimentation.
 21. The method of claim 20, wherein said positivesynergistic effect is chosen from surrogate endpoints associated withanimal experiment life extension, said surrogate endpoints being chosenfrom one or more surrogate endpoints selected from, but not limited to,the group consisting of reduced free T3, reduced fasting serum insulin,reduced fasting serum leptin, reduced basal body temperature, reducedserum triglycerides, enhanced beta fatty acid oxidation as indicated viareduced respiratory quotient, and surrogate endpoints associated withcaloric restriction such as the above and additional markers such asreduced mTOR and increased sirtuins in animals including humans.